Biosci. Biotechnol. Biochem., 71 (10), 2592–2595, 2007 Note 2-epi-Botcinin A and 3-O-Acetylbotcineric Acid from Botrytis cinerea Emi S AKUNO, Hiroko T ANI,* and Hiromitsu N AKAJIMAy Department of Agricultural Chemistry, Faculty of Agriculture, Tottori University, Tottori 680-8553, Japan Received May 30, 2007; Accepted June 19, 2007; Online Publication, October 7, 2007 [doi:10.1271/bbb.70334] Two metabolites, 2-epi-botcinin A and 3-O-acetylbotcineric acid, were isolated from Botrytis cinerea (AEM211). The former compound was new, and the latter was known but structurally revised by us. In a test for antifungal activity against Magnaporthe grisea, a pathogen of rice blast disease, 2-epi-botcinin A was 8 times less active than botcinin A (MIC 100 M), and the MIC value for 3-O-acetylbotcineric acid being 100 M. Key words: 2-epi-botcinin A; 3-O-acetylbotcineric acid; Botrytis cinerea; antifungal activity; Magnaporthe grisea Botrytis cinerea is a phytopathogen causing serious gray mold disease in many kinds of cultivated plants. The fungus produces many structurally diverse metabolites1–18) and is particularly well known as a producer of abscisic acid, a plant hormone.13) We have recently reported the isolation of new metabolites, botcinins A (1), B (2), C (3), D, E, and F, and the known metabolite, botcinolide, from an ethyl acetate extract of a strain of B. cinerea (AEM211) and their antifungal activity against Magnaporthe grisea, a pathogen of rice blast disease.19,20) In our previous study, the botcinins were shown to have a unique bicyclic unit and a fatty acyl portion, and we thoroughly reinvestigated the structure of botcinolide with a view to its structural relationship to botcinins. As a result, the structure of botcinolide (5) has been revised from a 9-membered macrolide to the seco acid of botcinin E (7), and botcinolide has been renamed botcinic acid. Homobotcinolide (6), a botcinolide analogue, has also been structurally revised to 8 and renamed botcineric acid.20) Our continuing search for new botcinins resulted in the isolation of a new botcinin and a known 3-Oacetylhomobotcinolide21) which was structurally revised to a botcineric acid analogue by precise interpretation of its spectroscopic data and its conversion to a known compound. We report in this paper the isolation and structure of 2-epi-botcinin A (4) and 3-O-acetylbotcineric acid (9) and describe their antifungal activity. Botrytis cinerea AEM 211, which had been isolated from a diseased strawberry in Tottori Prefecture,19) was y * O O CH3 H 1 3' O CH OAc 3 R1 CH3 CH3 H H 1: 2: 3: 4: HO HO H3C HO 5' 7' O CH3 3 R1 R3 1' 7 5 R2 OH O R2 H H CH3 CH3 R3 CH3 (CH2)2CH3 (CH2)2CH3 CH3 CH3 OH R O 5 7 3 1 H3C O CH3 O O R 5: CH3 6: (CH2)2CH3 CH3 HO HO CH3 1 O R1 7: H 8: H 9: Ac 3 OH O 5 O CH3 OR1 R2 1' 7 3' 5' 7' O CH3 R2 CH3 (CH2)2CH3 (CH2)2CH3 Fig. 1. Structures of Botcinins A (1), B (2), C (3), 2-epi-Botinin A (4), Botcinic Acid (7), Botcineric Acid (8), and 3-O-Acetylbotcineric Acid (9). cultured on a malt extract medium, without shaking, at 24
C for 14 days in the dark. The metabolites in the culture filtrate were extracted with ethyl acetate and separated into neutral and acidic fractions. The neutral fraction was purified by chromatographic separation to afford two anisaldehyde-positive compounds, 4 and 9, in respective yields of 1.1 and 0.7 mg/l. Compound 4 was obtained as an amorphous solid. The HRFABMS and NMR data for 4 afforded the To whom correspondence should be addressed. Tel/Fax: +81-857-31-5362; E-mail: nakajima@muses.tottori-u.ac.jp Present address: Yamada Bee Farm, Institute for Bee Products & Health, Kagamino-cho, Tomada-gun, Okayama 708-0393, Japan Botcinin Analogues Produced by Botrytis cinerea Table 1. 1 H- and 13 2593 C-NMR Data for Compounds 4 and 9 in CDCl3 4 Position C 1 2 2-CH3 3 4 4-CH3 5 6 6-CH3 7 8 8-CH3 10 20 30 40 50 60 70 80 90 100 CH3 CO 172.4 42.0 16.5 77.9 73.1 10.1 79.4 35.3 13.5 76.3 68.2 18.1 165.6 119.0 151.8 71.1 36.4 27.3 22.5 13.9 20.9 170.0 9 H (mult., J in Hz) C 2.61 (dq, J ¼ 7:8, 7.3) 1.43 (d, J ¼ 7:3) 4.96 (d, J ¼ 7:8) 1.33 3.71 2.11 1.07 4.52 3.68 1.07 (s) (d, J ¼ 11:2) (ddq, J ¼ 10:1, 11.2, 6.2) (d, J ¼ 6:2) (dd, J ¼ 10:1, 10.1) (dq, J ¼ 10:1, 6.2) (d, J ¼ 6:2) 6.06 (dd, J ¼ 1:6, 15.6) 7.01 (dd, J ¼ 4:6, 15.6) 4.33 (m) 1.53–1.65 (m) 1.24–1.39 (m) 1.24–1.39 (m) 0.91 (t, J ¼ 7:1) 2.11 (s) molecular formula of C22 H34 O8 . The 1 H-NMR and 13 CNMR data for 4 (Table 1) mostly agreed with those for botcinin C (3, C24 H38 O8 ), although two signals assignable to the methylene carbons of the acyl side chain were lost in the 13 C-NMR data for 4,19) indicating that compound 4 had two fewer methylenes than 3 in the acyl portion. The partial structure of CO–CH=CH– CH(OH)– in the acyl portion of 4 and E geometry of the double bond was confirmed by the 1 H-NMR data. The fragment ion assignable to the acyl portion was also observed at m=z 169 in the EIMS spectrum of 3 and at m=z 141 in that of 4. The relative stereochemistry of the ring portion of 4 was suggested to be identical to that of 3 by the NOE correlations. Irradiation of the 4-methyl protons caused enhancement in the resonances of H-2 and H-8, whereas no effect was apparent on H-5. The effect on H-6 by irradiating the 4-methyl protons was not clear due to overlapping of the acetyl methyl resonance. Irradiation of H-5 produced NOE in the resonances of H-3 and H-7. The optical rotation values for 3 and 4, ½ 25 D 28
and ½ 25 D 31
, respectively, were almost the same, indicating that the absolute stereochemistry of 3 and 4 was probably the same. Compound 4 differs from botcinin A (1) only by the stereochemistry at C-2 and thus is identical to 2-epibotcinin A. Compound 9 was isolated as a colorless oil. The molecular formula of 9 was deduced as C24 H40 O9 from NMR and FABMS data, which showed the ½M þ Naþ and ½M þ Hþ ions at m=z 495 and 473, respectively. The 1 H- and 13 C-NMR data for 9 (Table 1) were similar to those for botcinin B, C24 H38 O8 (2), except for some 172.9 40.0 17.3 77.8 79.8 14.8 71.1 37.0 14.0 75.7 69.5 17.5 165.7 119.0 151.8 71.1 36.7 25.2 29.1 31.7 22.5 14.0 20.9 172.5 H 3.09 (dq, J ¼ 1:6, 7.4) 1.28 (d, J ¼ 7:4) 4.95 (d, J ¼ 1:6) 1.40 3.11 1.95 1.00 4.50 3.77 1.17 (s) (d, J ¼ 10:8) (ddq, J ¼ 10:8, 10.8, 6.4) (d, J ¼ 6:4) (dd, J ¼ 9:9, 10.8) (dq, J ¼ 9:9, 6.0) (d, J ¼ 6:0) 6.06 (dd, J ¼ 1:6, 15.6) 6.99 (dd, J ¼ 4:6, 15.6) 4.34 (m) 1.60 (m) 1.37–1.43 (m) 1.25–1.34 (m) 1.25–1.34 (m) 1.25–1.34 (m) 0.88 (t, J ¼ 6:9) 2.23 (s) differences in chemical shifts. In the 13 C-NMR spectrum of 9, C-2, C-3, C-4, C-5, 2-CH3 , and 4-CH3 resonances were observed 2.8 ppm downfield, 3.5 ppm downfield, 4.6 ppm downfield, 7.4 ppm upfield, 7.1 ppm downfield, and 2.9 ppm downfield, respectively, compared to that of botcinin B. These shifts were due to the disappearance of the lactone ring. To verify this speculation, we treated 9 with acetic anhydride and pyridine to afford an acetylated and lactonized product, this being identical in all respects, including optical rotation, to the compound obtained by acetylating botcinin B. Thus, 9 was identified as 3-O-acetylbotcineric acid, and the absolute configuration was assigned as 2R, 3S, 4S, 5S, 6S, 7R, 8S, 40 S. The 1 H-NMR data for 9 agreed well with those reported for 3-O-acetylhomobotcinolide21) in the same solvent. 3-O-Acetylhomobotcinolide should be revised structurally to 3-O-acetylbotcineric acid. Compounds 4 and 9 were tested for their antifungal activity against Magnaporthe grisea. The MIC value for compound 4 was 800 mM, while those for botcinin A (1), B (2), and C (3) were 100 mM, 12.5 mM, and 100 mM, respectively.19) Compounds 4 and 3 are 2-epimers of 1 and 2, respectively. Compounds 2 and 3 each have a C10 acyl portion, while compounds 1 and 4 have a C8 acyl portion. Thus, in C-2, the R configuration produced enhancement of the antifungal activity compared with the S configuration, the length of the acyl chain also being important for the activity. The MIC value for compound 9, the seco acid of botcinin B, was 100 mM. We had previously reported the MIC value for botcinic acid (7) as 100 mM, but this did not show antifungal activity even at 800 mM in this study. This unexpected 2594 E. SAKUNO et al. result suggested a problem with the sample used in the assay. We therefore collected the NMR data for the sample and then confirmed the identity and purity of the sample. At the same time, the assay on the sample was repeated again and again, but no activity could be found, even at 800 mM. The reason why botcinic acid showed a 100 mM MIC value in the previous experiment is still not clear. The antifungal activity of 9 and botcinic acid (7) suggest that the length of the acyl chain and/or the acetoxyl group in C-3 in botcinic acid analogues was important for their activity. Many botcinic acid analogues need to be tested before determining the relationship between the antifungal activity and structure. Experimental General information. Optical rotation values were measured with a Horiba SEPA-200 polarimeter. UV spectra were recorded with a Hitachi U-2001 spectrophotometer, and IR spectra with a Jasco FT/IR 7000 spectrometer. NMR spectra were measured with a Jeol JNM-ECP 500 spectrometer. Chemical shifts were referenced to CDCl3 (H 7.26, C 77.0). Mass spectra were obtained with a Jeol AX505HA spectrometer (direct probe). p-Nitrobenzyl alcohol was the matrix used for FABMS, and the reaction gas for CIMS was isobutane. HPLC was carried out in a Cosmosil 5C18 AR column (Nacalai Tesque, 10  250 mm), using 75% MeOH in 1% AcOH as the eluent at a flow rate of 1.0 ml/min, with detection at 220 nm. Merck Kieselgel 60 F254 was used for TLC. The spots on TLC plates were detected by spraying the anisaldehyde-sulfuric acid reagent onto the plate and heating it at 110
C for 20 min. The spray reagent was prepared flesh before use by adding 1 ml of concentrated H2 SO4 to a solution of 0.5 ml of p-anisaldehyde in 50 ml of acetic acid. Fermentation and isolation. The fungus was grown without shaking at 24
C for 14 days in the dark in 500ml conical flasks (50) containing a liquid medium (200 ml/flask) composed of glucose (30 g/l), peptone (3 g/l), the extract from 50 g/l of malt, and tap water. Metabolites were extracted from the culture filtrate with EtOAc (3  10-liter) after adjusting the pH value to 2.0 with 6 M HCl. The EtOAc solution was washed with 1 M NaHCO3 (2  0:5 volume), dried over Na2 SO4 , and concentrated to dryness to give a residue (5.2 g). This residue was subjected to silica gel column chromatography (Daiso gel IR-60, 31  200 mm), with 1200 ml (240 ml  5) each of 10%, 20%, 30% and 40% acetone in n-hexane as the eluent. The third fraction (186 mg), eluted with 20% acetone in n-hexane, was purified by Sephadex LH-20 column chromatography (20  900 mm, MeOH). Five-milliliter portions of the eluate were collected. Fractions 12–33 were combined and purified further by HPLC to give compound 4 (11 mg, Rt 34 min). Fractions 1–3 (249 mg) of 30% acetone in nhexane were subjected to ODS flash column chromatography (Cosmosil 75C18 -PREP, 22  75 mm), with 150 ml (10 ml  15) each of 50%, 60%, 70%, and 80% MeOH as the eluent. Fractions 3–5 of 60% MeOH were combined and purified by HPLC to give compound 9 (7 mg, Rt 64 min). 2-epi-Botcinin A (4). Amorphous solid. ½ 25 D 31
(c 0.26, EtOH). UV max (EtOH) nm ("): 214 (12,700). IR (KBr) max cm1 : 3446, 2940, 2878, 1752, 1729. NMR data: see Table 1. FABMS m=z 427 ðM þ HÞþ . HRFABMS m=z ðM þ HÞþ : calcd. for C22 H35 O8 , 427.2332; found, 427.2334. 3-O-Acetylbotcineric acid (9). Colorless oil. ½ 25 D 5:20
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